This invention relates to chemically synthesized immunogens which generate antibodies specific for the activation peptide from prothrombin, F1.2. In another aspect, it relates to methods for detecting the presence and/or concentration of F1.2 in plasma.
Many proteins are synthesized in vivo as inactive precursors, or zymogens, which are subsequently converted to their active forms by limited proteolysis. This process of zymogen activation is a rate-limiting step in a variety of physiological processes which include blood coagulation. Blood coagulation proteins are xe2x80x9cactivatedxe2x80x9d by highly specific, limited peptide bond cleavage, which generate new carboxy and amino termini. The amino acid sequence within these termini is highly specific for a particular coagulation protein. These termini which are often located at the surface of the protein, show a relatively high conformational flexibility and are frequently good antigenic determinants.
Prothrombin is one of a series of proteins that participate in the blood clotting mechanism. Thrombin has many functions including catalyzing the conversion of fibrinogen to fibrin, and thus preventing the loss of blood from the vascular system of vertebrates. Prothrombin is proteolytically cleaved to form its active state thrombin; in this conversion the polypeptide fragment F1.2 is formed.
The detection of prothrombin activation products within whole blood or plasma has been attempted using immunological methods, but has been impaired because of the cross-reactions between the activation products and the parent molecule. Studies on radioimmunologic techniques for F1.2 in human plasma, have shown that there is significant cross-reactivity between F1.2 and related proteins such as F1, prothrombin and prethrombin-2. Cross-reactivities between the activation product and prothrombin, thus preclude the quantitation of this fragment in whole blood. In addition, the concentration of prothrombin is exceedingly high compared with the minute quantities of activation fragment that is expected to be formed in vivo.
The isolation of specific antibody population directed against cleavage fragments has allowed for the demonstration of small amounts of F1.2 in normal human plasma, even in the presence of normal prothrombin levels. Lau et al., The Isolation and Characterization of Specific Antibody Population Directed Against the Prothrombin Activation Fragments F2 and F1+2, 254 J. Bio. Chem. 8751 (1979). Additionally, Prothrombin catabolism has also been studied by screening urine for prothrombin derivatives. Bezeaud et al., Identification of Prothrombin Derivatives in Human Urine, 13 Thrombin Res. 551 (1978). Further studies by this group showed that in urine, where prothrombin is absent, it was possible to accurately quantitate F1 and F2 fragments. Bezeaud et al, Quantitation of Prothrombin Activiation Products in Human Urine, 58 Brit. J. Haemat. 597 (1984).
According to the present invention, highly specificxe2x80x94low affinity antibodies are generated which allow for the assay of F1.2 in bodily fluids that also contain prothrombin, or other plasma proteins. The antibodies are made using synthetic polypeptides which mimic the carboxy terminus of F1.2. The synthetic polypeptide of this invention elicited antibodies specific for F1.2, but which do not bind to intact prothrombin or other plasma proteins. This specificity is of particular importance in measuring F1.2 in plasma since prothrombin is normally present in a 1000-fold molar excess over F1.2.
Many different peptides were synthesized. The specific synthetic polypeptide used to raise antibodies with the highest specificity for F1.2 was found to have the following amino acid sequence: [CYS-GLY]-ASP-ARG-ALA-ILE-GLU-GLY-ARG-OH. Glycine and cysteine residues were added to permit convenient attachment of the synthetic polypeptide to a carrier protein. These additional amino acids (CYS-GLY), however, are not critical to this invention. This polypeptide-protein conjugate was used to elicit an immune response in laboratory animals.